Browsing by Author "Carpinteiro, Dina"
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- ABC system used as an add-on to clarify germline variants previously classified as VUS according to ACMG guidelinesPublication . Rodrigues, Pedro; Theisen, Patrícia; Silva, Catarina; Mendonça, Joana; Carpinteiro, Dina; Vieira, Luís; Gonçalves, JoãoThe increasing number of patients screened by NGS to identify germline variants associated with hereditary breast/ovarian cancer (HBOC) syndromes, is leading to a growing number of variants classified as Variants of Uncertain Significance (VUS) according to ACMG guidelines1. Since the ACMG system merges functional and clinical data into a one-dimensional system, it is not always clear how the classification was obtained. The ABC system (ABCs) of variant classification2 splits functional and clinical grading and aims to give a better guide to variant significance. The main goals of this work were i) to apply the ABCs to a group of previously classified ACMG-VUS and ii) to evaluate the potential clinical impact of this review/classification. Germline variants (36 - 29 missense, 1 synonymous and 6 intronic) detected in 5 genes (BRCA1, BRCA2, ATM, CHEK2, PALB2) previously classified as ACMG-VUS, were selected from our database of patients with HBOC, to be reclassified with the ABCs. Variant assessment included: query of clinical and population databases, literature and in silico tools (VEP, HSF, Alamut, Varsome). Eleven variants were classified as Class 0 (functional - fVUS); 17 as class E (functional - HFE (Hypothetical Function Effect), and 8 as Class D (functional - LFE (Likely Functional Effect). fVUS are not clinically graded. Considering that ACMG-VUS are not actionable, it is still an ongoing debate if they should be reported or not. Since the ACMG merges functional and clinical data, it might be difficult for clinicians to understand how VUS classification is achieved. The ABCs allowed us to distinguish between VUS classified due to lack of data from those that might have a functional impact. Class 0 variants (11) should not be reported and class E (17) reporting is optional. The use of ABCs highlighted 8 variants (class D) which might be a susceptibility factor with functional impact and should be reported. Functional and segregation studies are of major importance to clarify the clinical significance of these variants. 1-PMID: 25741868. 2-PMID: 33981013. Support: FCT/MCTES, ToxOmics and Human Health (UIDB/00009/2020). GenomePT(POCI-01-0145-FEDER-022184).
- Accreditation under the International Standard ISO 15189: Experience of a Genetics Laboratory in DNA SequencingPublication . Silva, Catarina; Cardoso, Ana; A Sampaio, Daniel; Carpinteiro, Dina; Mendonça, Joana; Duarte, Sílvia; Barreiro, Paula; Torgal, Helena; Isidro, Glória; Vieira, LuísIntroduction: Health care is to some extent influenced by the results of laboratory tests. In order to provide the best care for the patient, laboratories must seek to achieve high levels of quality and competence. International Standard ISO 15189 specifies these requirements and may be used by laboratories to perform accredited genetic tests of materials derived from the human body. Here we describe the procedures to establish Accreditation of DNA sequencing in our laboratory and the first Accreditation of its kind in Portugal. Methods: Our laboratory started to prepare to comply with ISO 15189 Accreditation requirements for DNA sequencing in 2010. Documents describing administrative and technical procedures of the sequencing workflow including sample registries, laboratory protocols, operation and maintenance of equipments, as well as preparation and use of reagents were produced. Regular examination of laboratory equipments by an external entity was implemented to confirm compliance with working requirements. Requisites for personnel training and demonstration of competence were also implemented. The laboratory participated regularly in the DNA sequencing scheme organized by the European Molecular Genetics Quality Network (EMQN). Results: The laboratory obtained formal recognition by Instituto Português de Acreditação (IPAC) in May 2014. A maximum genotyping score for DNA sequencing has been obtained in the external quality assessment scheme since 2010. Sequencing quality measured in terms of the quality read overlap metrics is currently of approximately 96% according to the EMQN scheme. The laboratory processes and analyzes an average of 28.750 samples per year. Discussion: Accreditation of a genetic test under ISO 15189 is a highly demanding and laborious task for a genetic laboratory. However, it is an important step in order to guarantee the highest quality and reproducibility of genetic test results.
- Avaliação do desempenho de uma core-facility de sequenciação genómica especializada em saúde públicaPublication . Vieira, Luís; Silva, Catarina; Duarte, Sílvia; Mendonça, Joana; Carpinteiro, Dina; Sampaio, Daniel A.; Ferrão, José; Santos, Daniela; Machado, Miguel; Isidro, Joana; Barreiro, Paula; Isidro, GlóriaA Unidade de Tecnologia e Inovação (UTI) do Departamento de Genética Humana foi criada em 2009 pelo despacho normativo n.º 15/2009. Apesar de estar integrada num departamento técnico científico, esta unidade constituiu-se desde logo como core-facility de sequenciação genómica do Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA). Este papel envolve uma gestão contínua de prioridades dos serviços a prestar aos utilizadores, no âmbito da resposta a diferentes problemas de saúde pública, aliada a uma preocupação permanente com a qualidade dos resultados e os tempos de resposta. Neste trabalho, apresentamos os resultados da avaliação do desempenho da UTI, desde a introdução da tecnologia de Next-Generation Sequencing (NGS) em 2013, em termos de: (i) métricas de produção da Unidade, (ii) impacto dos resultados publicados no âmbito de colaborações científicas com os grupos de investigação do INSA ou de entidades externas e de (iii) avaliação dos serviços através de um inquérito dirigido aos utilizadores. Até final de 2021, o número de ensaios de NGS e de citações dos trabalhos publicados cresceram, por ano, 39% e 61%, respetivamente. Os utilizadores avaliaram de forma muito positiva os serviços prestados pela UTI em 2021. Globalmente, estes resultados demonstram que o modelo de trabalho de "core- -facility" exercido pela UTI é uma mais-valia na resposta aos problemas da saúde pública em Portugal.
- BRCA1 and BRCA2 variants identified in patients with a personal/familial history of hereditary breast/ovarian cancers and other hereditary cancer syndromes: challenges related with variants of uncertain significancePublication . Rodrigues, Pedro; Theisen, Patrícia; Silva, Catarina; Carpinteiro, Dina; Mendonça, Joana; Vieira, Luís; Gonçalves, JoãoIntroduction: Screening for BRCA1 and BRCA2 variants (Vs) in patients with Hereditary Breast/Ovarian Cancer (HBOC) or other Hereditary Cancer Syndromes (HCS) is performed using next-generation sequencing (NGS), allowing detection of a high number and types of Vs. The growing use of PARP inhibitors (PARPi) in the treatment of patients with homologous recombination-deficient tumors contributes to an increasing number of patients being screened for BRCA Vs even when family history of HBOC/HCS is absent. These approaches result in a growing number of identified Vs that need to be classified. The goals of this study, apart from identifying pathogenic and likely pathogenic Vs, were to identify uncertain significance Vs (VUS) and bring to discussion their uncertainties and impact on patients and family members. Methodology: BRCA1 and BRCA2 were analyzed in 207 patients mainly with HBOC/HCS, using TruSight® Cancer and MiSeq. Annotation was performed with Variant Interpreter, VEP, HSF, IGV, Alamut and Varsome. Vs were divided in 3 groups (G) according to allele frequency (AF) in population databases (G1:AF>5%, G2:1%≤AF≤5% and G3:AF<1%) and classified according to ACMG-AMP guidelines. Results: In BRCA1 and BRCA2, 45 and 96 Vs were detected, respectively. While in BRCA1 G3, we detected 6 pathogenic (P) Vs and 9 VUS, in BRCA2 G3, we found 9P Vs, 2 Likely Pathogenic (LP), and 15 VUS. We highlight that in G3, VUS were more frequent than P and LP Vs. Discussion: Among G3, 28% of BRCA1 and 25% of BRCA2 Vs were VUS. VUS give rise to difficulties related to management of patients and families. Functional studies of missense or putatively affecting splicing VUS are of major importance to assess their biopathologic impact, as some of them may be hypomorphic and reclassified as P/LP. Accordingly, some VUS may have impact in therapeutic decisions (e.g. PARPi) as well as in patient’s cancer-risk management protocols, including appropriate genetic counselling and VUS screening in selected family members. We predict that new challenges related to VUS will emerge.
- Comparison of Multi-locus Genotypes Detected in Aspergillus fumigatus Isolated from COVID Associated Pulmonary Aspergillosis (CAPA) and from Other Clinical and Environmental SourcesPublication . Morais, Susana; Toscano, Cristina; Simões, Helena; Carpinteiro, Dina; Viegas, Carla; Veríssimo, Cristina; Sabino, RaquelBackground: Aspergillus fumigatus is a saprophytic fungus, ubiquitous in the environment and responsible for causing infections, some of them severe invasive infections. The high morbidity and mortality, together with the increasing burden of triazole-resistant isolates and the emergence of new risk groups, namely COVID-19 patients, have raised a crescent awareness of the need to better comprehend the dynamics of this fungus. The understanding of the epidemiology of this fungus, especially of CAPA isolates, allows a better understanding of the interactions of the fungus in the environment and the human body. Methods: In the present study, the M3 markers of the STRAf assay were used as a robust typing technique to understand the connection between CAPA isolates and isolates from different sources (environmental and clinical-human and animal). Results: Of 100 viable isolates that were analyzed, 85 genotypes were found, 77 of which were unique. Some isolates from different sources presented the same genotype. Microsatellite genotypes obtained from A. fumigatus isolates from COVID+ patients were all unique, not being found in any other isolates of the present study or even in other isolates deposited in a worldwide database; these same isolates were heterogeneously distributed among the other isolates. Conclusions: Isolates from CAPA patients revealed high heterogeneity of multi-locus genotypes. A genotype more commonly associated with COVID-19 infections does not appear to exist.
- Detection of somatic mutations in Wilms tumours using gene panel sequencingPublication . Silva, Catarina; Carpinteiro, Dina; Ataíde Sampaio, Daniel; Vieira, LuísIntroduction: Wilms tumour (WT) is an embryonal kidney neoplasia in which the causative mutations are largely unknown. However, approximately one third of patients display somatic mutations in WT1, CTNNB1, TP53 and/or WTX genes, prompting the design of molecular tests to determine the mutational profile of each patient. In this work we describe a novel molecular assay based on next-generation sequencing (NGS) technology which we used to identify mutations in 36 Portuguese WT patients. Methods: Design Studio (Illumina) was used to create a sequencing panel of 83 PCR amplicons covering 12.306 bases of exonic sequences of WT1, CTNNB1, TP53 and WTX genes. Amplicons were prepared from tumour and matched peripheral blood DNA samples (n=73) using a TruSeq Custom Amplicon kit (Illumina). Libraries were sequenced on a MiSeq instrument using paired-end 250 bp reads. Sequence reads were aligned to hg19 human genome reference sequence using MiSeq Reporter software (Illumina). Variants were annotated using publicly available databases. Results: Data analysis of the constitutional DNA of WT patients showed the existence of 31 germline variants, including 9 variants not described in the human dbSNP database. Comparison of matched tumour samples revealed the presence of 14 putative mutations in 12 patients. The mutations included WT1 (n=3), CTNNB1 (n=4), WTX (n=5) and TP53 (n=2). In one patient, concomitant WT1 and CTNNB1 mutations were found. Comparison of results with previous Sanger sequencing data for WT1 and CTNNB1 in the same samples confirmed 5 out of 7 mutations detected by NGS in which the mutated allele frequency was above 20%. Discussion: We conclude that gene panel sequencing is a fast and sensitive molecular assay for identification of recurrent somatic mutations in WT. However, because two thirds of patients lack known mutations, other NGS-based approaches such as exome sequencing may be fruitful to identify novel mutations in WT.
- Development and testing of microsatellite loci for the study of population genetics of Ixodes ricinus Linnaeus, 1758 and Ixodes inopinatus Estrada-Peña, Nava & Petney, 2014 (Acari: Ixodidae) in the western Mediterranean regionPublication . Velez, Rita; De Meeûs, Thierry; Beati, Lorenza; Younsi, Hend; Zhioua, Elyes; Antunes, Sandra; Domingos, Ana; Ataíde Sampaio, Daniel; Carpinteiro, Dina; Moerbeck, Leonardo; Estrada-Peña, Agustin; Santos-Silva, Maria Margarida; Santos, Ana SofiaIxodes ricinus is an important vector of several human and veterinary infectious agents. Its wide geographical distribution and permissive feeding behaviour have prompted earlier studies on its population genetics. Results were, nevertheless, not conclusive. Furthermore, no research has fully focused on the south-western distribution range of I. ricinus, where exchanges between European and North African populations are more likely to occur. The presence of an additional species, Ixodes inopinatus, in the area further confuses the topic, as the two species are hard to differentiate morphologically. The present work describes the testing of microsatellite markers previously described for I. ricinus using Portuguese and Tunisian tick populations of both species. In addition, new microsatellite loci were developed to complement the available marker toolbox. Loci showed different amplification successes across subpopulations, with Tunisian DNA less readily amplified. Altogether, 15 loci were considered suitable for genetic analyses of Portuguese subpopulations, 10 for Tunisian samples, and seven, common to both populations, were considered to be informative at the inter-continental level. A preliminary analysis of both datasets revealed two isolated populations, which can correspond to two different species. Furthermore, Tunisian specimens identified by sequencing of 16S rDNA as having I. ricinus or I. inopinatus sequence profiles all clustered together in one single population using the proposed microsatellites. This confirms that taxonomic decisions based only on 16S rRNA gene sequencing can be misleading. The application of the proposed set of microsatellite markers to a larger sample, representative of the south-western Ixodes’ distribution range, will be crucial to clarify the distribution of both species.
- Estudo mutacional em tumor de Wilms por sequenciação de nova geraçãoPublication . Silva, Catarina; Carpinteiro, Dina; Vieira, LuísO Tumor de Wilms (TW) é o tumor renal mais comum da infância, com uma incidência de 1 em ~10000 crianças. Esta patologia é de etiologia genética complexa e diversificada. No entanto, cerca de um terço dos doentes apresenta mutações somáticas associadas aos genes WT1, CTNNB1, TP53 e/ou AMER1. Assim, foi desenvolvido um painel de amplicões destes 4 genes para a identificação de mutações num grupo de doentes portugueses com TW, através de uma metodologia baseada na sequenciação de nova geração. As bibliotecas de DNA foram preparadas a partir de amostras de sangue periférico e tumor de 36 doentes com TW e sequenciadas no MiSeq. Foram identificadas alterações somáticas em 7 dos 36 (19,4%) doentes estudados. Conclui-se que a sequenciação de um painel de genes é um método rápido para a deteção de mutações somáticas quando desenhado com cuidado de forma a serem evitados problemas de perda de cobertura.
- Genetic testing for germline variants in homologous recombination repair genes, other than BRCA1 and BRCA2, in patients with suspected hereditary cancer syndromesPublication . Arnaut, Daniela; Rodrigues, Pedro; Theisen, Patrícia; Carpinteiro, Dina; Vieira, Luís; Gonçalves, JoãoHomologous recombination repair (HRR) is the cellular mechanism for error-free repair of DNA double-strand breaks. Pathogenic germline variants in BRCA1 and BRCA2 lead to HRR deficiency associated with breast, ovarian, prostate, pancreatic cancers and are sensitive to PARP inhibitors (PARPi). Defects in HRR genes beyond BRCA1/2 could also result in HRR deficiency and sensitize the tumor to PARPi, thus expanding the subset of patients that can benefit from these targeted therapy cancer drugs. We studied 56 DNA samples obtained from patients with personal and family history of cancer. Genes involved in HRR (ATM, BAP1, BLM, BRIP1, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, NBN, PALB2, RAD51C, RAD51D) were analysed by NGS using TruSight® Hereditary Cancer. Sequence alignment and annotation included DRAGEN Enrichment and Variant Interpreter - Illumina®. Variant classification, according to ACMG-AMP 1, was based on VEP, HSF, Alamut, VarSome and several databases (ex. HGMD, gnomAD, dbSNP). Variants of uncertain significance (VUS) were also classified with the stepwise ABC system2. All pathogenic/likely pathogenic SNVs and CNVs were confirmed by Sanger sequencing or MLPA. We identified 156 SNVs and one CNV, of these 125 were benign/likely benign. Seven clinically actionable variants were found in 10.7% of the patients: 3 pathogenic variants in FANCA, FANCD2 and FANCI give rise to premature stop codons and one pathogenic CNV in FANCA (deletion of exons 38 and 39); 2 likely pathogenic variants in BLM and FANCI affecting splicing and one frameshift in FANCG. Classification of 18 VUS with the ABC system resulted in: 8 class 0 (normal finding), 7 class E (potential interest) and 3 class D (low penetrance) variants. In addition, 7 SNVs were classified as hypomorphic alleles. This study confirmed: i) the importance of extending the molecular study beyond BRCA1/2 to other genes involved in HRR, ii) some variants require functional/family studies to establish their pathogenicity, and iii) these genes could potentially be considered for specific and clinical studies involving PARPi therapy.
- Genome-scale analysis of the non-cultivable Treponema pallidum reveals extensive within-patient genetic variationPublication . Pinto, Miguel; Borges, Vítor; Antelo, Minia; Pinheiro, Miguel; Nunes, Alexandra; Azevedo, Jacinta; Borrego, Maria José; Mendonça, Joana; Carpinteiro, Dina; Vieira, Luís; Gomes, João Paulo.Insights into the genomic adaptive traits of Treponema pallidum, the causative bacterium of syphilis, have long been hampered due to the absence of in vitro culture models and the constraints associated with its propagation in rabbits. Here, we have bypassed the culture bottleneck by means of a targeted strategy never applied to uncultivable bacterial human pathogens to directly capture whole-genome T. pallidum data in the context of human infection. This strategy has unveiled a scenario of discreet T. pallidum interstrain single-nucleotide-polymorphism-based microevolution, contrasting with a rampant within-patient genetic heterogeneity mainly targeting multiple phase-variable loci and a major antigen-coding gene (tprK). TprK demonstrated remarkable variability and redundancy, intra- and interpatient, suggesting ongoing parallel adaptive diversification during human infection. Some bacterial functions (for example, flagella- and chemotaxis-associated) were systematically targeted by both inter- and intrastrain single nucleotide polymorphisms, as well as by ongoing within-patient phase variation events. Finally, patient-derived genomes possess mutations targeting a penicillin-binding protein coding gene (mrcA) that had never been reported, unveiling it as a candidate target to investigate the impact on the susceptibility to penicillin. Our findings decode the major genetic mechanisms by which T. pallidum promotes immune evasion and survival, and demonstrate the exceptional power of characterizing evolving pathogen subpopulations during human infection.